A physical map of the human genome in terms of a set of overlapping yeast artificial chromosome (YAC) clones is now nearing completion (Chumakov, et al.; Hudson, et al., 1995). One of the necessary prerequisites to initiating a large scale sequencing effort will be to convert the YAC contig(s) into a minimum tiling path of cosmid or PAC clones. The major objective of this project is to streamline the generation of sequencing substrates and to minimize downstream sequencing redundancy by identifying cosmid and PAC clones using multiplex array screening and by using novel optical imaging techniques for constructing contig maps and identifying those clones which exhibit a minimal sequence overlap. The specific goals are: 1) To develop a procedure for screening gridded arrays of cloned DNA with up to 31 probes simultaneously, each identifiable by a unique spectral signature based on fluor- composition, using an epifluorescence microscope based array scanner. 2)To construct a high resolution cosmid and PAC contig map covering the entirety of human chromosome 12. (130Mb). Cosmid and PAC clones, corresponding to a minimal tiling path of nonchimeric YAC clones previously characterized, will be identified by conventional library screening and by multiplex fluorescence imaging. These clones will be ordered and their overlap ascertained by multicolor FISH to YAC DNA that has either been stretched on a glass slide or immobilized in an oriented fashion and extended in a mild electric field. Six or more cosmid or PAC clones, each labeled with a different fluorophores or combinination of fluorophores, will be hybridized simultaneously. 3) To continue to add new markers to our existing YAC contig map and our proposed cosmid(PAC maps of chromosome 12. Our current map consists of 1100 YACs and contains 1000 markers, approximately 1 per 130Kb. These include highly polymorphic genetic markers, monomorphic and anonymous markers as well as genes and ESTs. In order to increase the utility of our map until the complete sequence is obtained, we will use PCR screening and multiplex hybridization to arrayed ESTs to incorporate at least 1000 new markers into the map over the next three years.